1. Field of the Invention
This invention is related to the field of probe-based detection, analysis and quantitation of individual human chromosomes X, Y, 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 16, 17, 18 and 20 as well as 13/21 as a pair using detectable non-nucleic acid probes.
2. Description of the Related Art
Nucleic acid hybridization is a fundamental process in molecular biology. Probe-based assays are useful in the detection, identification, analysis and quantitation of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from bacteria, eucarya, fungi, virus or other organisms and they are also useful in examining samples for genetically-based disease states or clinical conditions of interest.
Chromosome disorders comprise a significant number of genetic diseases. For example, approximately 16 percent of recessive disorders are the result of X-linked genetic defects for which no specific diagnostic procedure is presently available (See: Delhanty et al. Human Mol. Genetics. 2: 1183-1185 (1993)). It has been estimated that detectable chromosomal abnormalities occur with a frequency of one in every 250 births (See: Epstein, The consequency of chromosome imbalance: principle, mechanism and models, Cambridge University Press, 1986; Lubs et al., Science, 169: 495-497 (1970); and Jacobs, Am. J. Epidemiol. 105: 180-191 (1970)). Abnormalities that involve the deletion or addition of chromosomal materials after the genetic balance of an organism has been determined can lead to serious mental or physical disease and even death. With the arrival, acceptance and rapid proliferation of in-vitro fertilization (IVF), research into methods and compositions suitable for the examination of the chromosomes of ova, spermatozoa, embryo and blastomeres have become commonplace. For example, recent reports have shown increased incidence of hyperhaplloid (24/XY) spermatozoa in males with 46 XY/47 XXY karyotypes (See: Cozzi et al, Hum. Genet., 93: 32-34 (1994); Chevret et al. Hum. Genet. 97: 171-175 (1996); Martini et al., Human Reproduction, 11: 1638-1643 (1996) and Estop et al., Human Reproduction, 13: 124-127 (1998)). For families affected by sex linked disorders, preimplantation diagnosis (PID) is essential to insure that the fetus is not affected. Thus, the examination of blastomeres for sex determination and chromosome disorder prior to implantation has become a routine part of the IVF processes since implantation of chromosomally defective embryos will result in either miscarriage or in the birth of an infant having a genetic defect (See: Harper, Journal of Assisted Reproduction and Genetics, 13: 90-95 (1996).
Pioneering work directed to preimplantation and prenatal sex determination was performed by Handyside and his colleagues (See: Handyside et al., The Lancet, 347-349 (February, 1989) and Handyside et al., Nature, 344: 768-770 (1990)). Handyside et al. used PCR to amplify repetitive satellite sequences of the Y-chromosome. This method was at first very attractive since results could be rapidly obtained (approximately 3 hours). Speed is a critical factor in preimplantation diagnosis since implantation can only occur within a short time when the female uterus is suitable to impregnation. Because only the Y-chromosome was detected, the PCR method developed by Handyside et al. predicted a male child when amplification occurred and a female child when no amplification occurred. However, this method had a rather high incidence of misdiagnosis of prenatal and preimplantation sex determination (See: Kontogianni et al., Preimplantation Genetics, Plenum Press, New York, pp. 139-145).
Though improvements were made to the PCR technique to allow for the simultaneous and more accurate detection of X and Y chromosomes (See: Chong et al., Hum. Mol. Genetics, 2: 1187-1191: (1993) and Strom et al., J. of in Vitro Fertilization and Embryo Transfer, 8: 225-229 (1991)), chromosomal disorders such as aneuploidy (including XXY and XYY karyotypes) and polyploidy were not detectable by improved PCR methods since the methods could not quantitate X and Y chromosomes but merely determine their presence or absence. Furthermore, PCR techniques are highly susceptible to misdiagnosis caused by small amounts of foreign (contaminating) DNA (See: Harper et al., Hum. Reproduction, 9: 721-724 (1994)).
Given the limitations of PCR, particularly with regard to misdiagnosis or non-diagnosis of aneuploidy and polyploidy conditions, in-situ hybridization (ISH), and particularly fluorescence in-situ hybridization (FISH), has become another used and often preferred method for the analysis of cells, tissues (including bone marrow), spermatozoa, ova, blastomeres, oocysts, buccal cells and chorinic ville. ISH and FISH can be used to examine both metaphase chromosome spreads and interphase nuclei. Because intracelluar chromosomes are routinely visualized (examined) within the nuclei or metaphase condition, the exact number of chromosomes per cell can be quantitated. Therefore, abnormal conditions such as aneuploidy and polyploidy are easily diagnosed. While FISH techniques have become established in clinical and medical applications utilizing nucleic acid probes, typically its Achilles Heal has been that the procedure is often slow to yield results as compared with PCR techniques.
Long arrays of tandemly repeated satellite DNAs are known to exist in the human genome and can generally be organized into distinct classes (See: Greig et al., Am. J. Hum. Genet., 45: 862-872 (1989). However, subsets of the satellite DNA classes appear to have evolved such that they are largely specific to the chromosome of origin. Thus, alpha satellite DNAs provide chromosome specific markers that can be used as a basis of individual chromosome identification. However, there is a possibility that these markers exist in low abundance on other chromosomes and this can lead to detectable cross reactions. (See: Greig et al., Am. J. Hum. Genet., 45: 862-872 (1989) at p. 865, col. 2, Ins. 6-15).
ISH and FISH based chromosome analysis is typically performed using DNA probes that are greater than 100 bp in length (often greater than a kb in length), and that typically have multiple labels and that are directed to target sequences of alpha satellite DNA of the chromosome sought to be detected. These probes are typically generated by digestion of naturally occurring DNAs (nick translation) or by enzymatic synthesis using naturally occurring DNA as a template. Thus, the probes are typically a heterogeneous population of numerous fragments, the exact composition of which varies substantially from preparation to preparation. Consequently, the performance of the probes will typically vary substantially from preparation to preparation.
Particularly when utilized in the same assay under a single set of stringency conditions, these nucleic acid probes (composition of nucleic acid fragments) may exhibit some cross reaction to other chromosomes of the sample (See: Matera et al., Genomics, 18: 729-731 (1993)). Cross reaction is at least partially the result of the strong sequence homology within the classes of alpha satellite DNA and thereby requires that the assay exhibit a high degree of discrimination for long DNA probes under preset conditions of stringency. Thus, typical oligonucleotide probes can exhibit cross reaction at, what is commonly referred to as, both low and even high stringency conditions. Since cross hybridization occurs under conditions of both low and high stringency, the signal to noise ratio is poor for these assays regardless of the nature of the stringency conditions. This is particularly disadvantageous for multicolor analysis wherein different fluorophores can exhibit different efficiencies for signal generation. Cross reaction can also be particularly disadvantageous in an assay which is automated since these processes often will mis-call weak signals as false positives.
A commonly used method for reducing cross reaction caused by non-specific hybridization in in-situ hybridization assays involves the use of “blocking nucleic acid” (See: Gray et al., U.S. Pat. No. 5,447,841). Common sources of blocking nucleic acid can include enzymatically digested DNA, salmon sperm DNA as well as other natural sources of heterogenous nucleic acid. Similarly, synthetic oligonucleotides can be used to reduce the binding of probes to non-target sequences though this methodology does not appear to have been utilized in chromosome analysis (See: Arnold et al., U.S. Pat. No. 5,434,047). Likewise, PNA probes have been used to suppress the binding of detectable probes to non-target sequences though again this methodology has not been previously applied to chromosome analysis (See: WIPO Patent Application: WO98/24933).
As applied to the analysis of chromosomes X and Y, ISH and FISH techniques have typically employed DNA probes that have multiple labels and that are typically greater than 100 base pairs (bp) in length and that are prepared by nick translation of cloned DNA greater than 1 kilobase (kb) in length (See: Chevet et al, Hum. Genet., 97: 171-175 (1996)). The most commonly used probes for analysis of sex disorders appear to be commercially available sets of probes CEP X/Y/18/13/21 (See: Munne et al., Human Reproduction, 8: 2185-2191 (1993)) that are available from Vysis, (formerly Imagenetics) or similar DNA probes that are available from Oncor (See: Martini et al., Human Reproduction, 11: 1638-1643 (1996)). Generally, the DNA probes for the analysis of X and Y are directed to target sequences of alpha satellite DNA of the chromosome sought to be detected.
As applied to the analysis of human chromosome 2, ISH and FISH techniques have typically employed nick translated DNA probes that have multiple labels and that are greater than 100 base pairs (bp) in length (See: Haaf et al., Genomics, 13: 122-128 (1992) referencing Greig et al., Am. J. Hum. Genet., 45: 862-872 (1989). Similarly, the FISH analysis of human chromosome 10 was performed using nick translated DNA probe or approximately 175 bp (See: Howe et al., Hum. Genet. 91: 199-204 (1993). Methods and sequence information suitable for producing, by digestion of chromosomal DNA or by nick translation, long DNA probes suitable for the analysis of human chromosome 6 can be found in Jabs et al., Am. J. Hum. Genet. 41: 374-390 (1997).
As applied to the analysis of chromosome 16, ISH and FISH techniques have typically employed nick translated DNA probes that have multiple labels and that are greater than 100 base pairs (bp) in length (See: Greig et al., Am. J. Hum. Genet., 45: 862-872 (1989) and Stallings et al., Genomics, 13: 332-338 (1992)). However, Stallings et al., did utilize a synthetic 35-mer for the identification and characterization of CH16LAR (lies on chromosome 16) sequences though this was not an ISH or FISH assay (See: Stallings et al., Genomics, 13: 332-338 (1992) at the section entitled “Identification And Characterization Of CH16LAR Sequences” beginning p. 336, col. 1). A PCR assay has been developed to assay chromosome 16 for loss of heterozygosity though this is not a probe-based assay (See: Kihana et al., Jpn. J. Cancer Res., 87: 1184-1190 (1996). Analysis of alpha satellite DNA of chromosomes 17 and 18 have utilized DNA probes of hundreds to thousands of nucleotides in length (See: Waye et al, Molecular and Cellular Biology, 6: 3156-3165 (1986) and Alexandrov et al., Genomics 11: 15-23 (1991), respectively).
The methods thus far described all relate to the analysis of chromosomes X, Y, 1, 2, 6, 10 16, 17 or 18 using conventional nucleic acid based assay formats. However, the FISH analysis of human chromosome 5 has been described using a 30-mer synthetic oligonucleotide probe (See: Matera et al., Genomics, 18: 729-731 (1993) directed to the centromeric region. This is the only example, of which Applicant is aware, of using a synthetic nucleic acid in a FISH format to identify a human chromosome.
Despite its name, Peptide Nucleic Acid (PNA) is neither a nucleic acid, a peptide nor is it even an acid. Peptide Nucleic Acid (PNA) is a non-naturally occurring polyamide that can hybridize to nucleic acid (DNA and RNA) with sequence specificity (See U.S. Pat. No. 5,539,082 and Egholm et al., Nature 365:566-568 (1993)). Being a non-naturally occurring molecule, unmodified PNA is not known to be a substrate for the enzymes that are known to degrade peptides or nucleic acids. Therefore, PNA should be stable in biological samples, as well as have a long shelf-life. Unlike nucleic acid hybridization that is very dependent on ionic strength, the hybridization of a PNA with a nucleic acid is fairly independent of ionic strength and is favored at low ionic strength, conditions that strongly disfavor the hybridization of nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567). The effect of ionic strength on the stability and conformation of PNA complexes has been extensively investigated (Tomac et al., J. Am. Chem. Soc. 118: 5544-5552 (1996)). Sequence discrimination is more efficient for PNA recognizing DNA than for DNA recognizing DNA (Egholm et al., Nature, at p. 566). However, the advantages in point mutation discrimination with non-nucleic acid probes, as compared with DNA probes, in a hybridization assay, appears to be somewhat sequence dependent (Nielsen et al., Anti-Cancer Drug Design 8:53-65, (1993) and Weiler et al., Nucl. Acids Res. 25: 2792-2799 (1997)).
Though they hybridize to nucleic acid with sequence specificity (See: Egholm et al., Nature, at p. 567), PNAs have been slow to achieve commercial success at least partially due to cost, sequence specific properties/problems associated with solubility and sell-aggregation (See: Bergman, F., Bannwarth, W. and Tam, S., Tett. Lett. 36:6823-6826 (1995), Haaima, G., Lohse, A., Buchardt, O. and Nielsen, P. E., Angew. Chem. Int. Ed. Engl. 35:1939-1942 (1996) and Lesnik, E., Hassman, F., Barbeau, J., Teng, K. and Weiler, K., Nucleosides & Nucleotides 16:1775-1779 (1997) at p 433, col. 1, ln. 28 through col. 2, In. 3) as well as the uncertainty pertaining to non-specific interactions that might occur in complex systems such as a cell (See: Good, L. et al., Antisense & Nucleic Acid Drug Development 7:431-437 (1997)). Consequently, their unique properties clearly demonstrate that PNA is not the equivalent of a nucleic acid in either structure or function. Thus, PNA probes need to be evaluated for performance and optimization to thereby confirm whether they can be used to specifically and reliably detect a particular nucleic acid target sequence, particularly when the target sequence exists in a complex sample such as a cell, tissue or organism.
PNA probes have been demonstrated as being useful for the detection of rRNA in ISH and FISH assays (See: WO95/32305 and WO97/18325). PNA probes have also been used in the analysis of mRNA (e.g. Kappa Light Chain), viral nucleic acid (e.g. human papillomavirus) and the analysis of centromeric sequences in human chromosomes. Importantly, a PNA probe has also been used to detect human X chromosome specific sequences in a PNA-FISH format (See: WO97/18325, now U.S. Pat. No. 5,888,733). The ISH based analysis of eukaryotic chromosomes and cells using polyamide nucleic acids has also been suggested (See: U.S. Pat. No. 5,888,734).
The analysis of the telomere length of human chromosomes has been demonstrated using PNA probes in a FISH assay (See: Lansdorp et al., Human Mol. Genetics, 5: 685-691 (1996) as well as WO97/14026). Telomere length was measurable since the intensity of fluorescence from the terminus of the chromosome was shown to be proportional to the number of hybridized PNA probes and therefore proportional to the length of the telomere.
Similarly, the analysis of trinucleotide repeats in chromosomal DNA using appropriate PNA probes has been suggested (See: WO97/14026). Subsequently, DNA and PNA probes were used to examine cells for genetic defects associated with expansion of trinucleotide repeats which manifest as the disease known as human myotonic dystrophy (See: Taneja, Biotechniques, 24: 472-476 (1998)). The molecular basis of myotonic dystrophy (DM) is an extreme expansion of CTG repeat sequences in the 3′-UTR of the transcripts for the myotonin protein kinase (DMPK) gene. The severity and age of onset of this disease is known to be proportional to the length of the repeat expansion. The intensity of fluorescence from PNA probes hybridized to the targeted repeats could be used to quantify the length of the expansion repeat. This suggests the possibility of expansion repeat quantitation in a manner useful to confirm the diagnosis of genetic disease and possibly quantifying the age of onset and anticipated severity of the disease.
The Applicant is unaware of any previously described use of peptide nucleic acid (PNA) probes for the detection, identification or enumeration of chromosomes Y, 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 20 and 21.
In summary, any methods, kits or compositions that could improve the specificity, sensitivity and reliability of probe-based assays for the detection of chromosomes X, Y, 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 18, 20 and 21 would be a useful advance in the state of the art particularly where the methods were uniformly applicable to probes of all or substantially all sequence variations. Moreover, the methods, kits or compositions would be particularly useful if they could provide for the rapid, reliable, accurate, sensitive and automated multiplex analysis of samples for the presence, absence or number of chromosomes X, Y, 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 16, 17, 18 and 20, as well as 13/21 as a pair. It would be most useful if the methods, kits and compositions were suitable for the simultaneous analysis of some or all of the human chromosomes in the same assay.